Platelets are central to hemostasis because they respond to vascular damage and secrete a variety of granule cargo molecules, which are critical to thrombosis and its normal, as well as pathogenic, sequellae. This property makes platelets a target for modulation of hemostasis but it also raises questions about how these anucleated cell fragments function. Are platelets an active participant specifically releasing context-appropriate material or are they random delivery devises? The overall goal of this proposal is to understand the process of platelet secretion at the cellular and molecular level. The first Aim addresses one of the most important questions facing the field. Is platelet secretion thematically responsive? The experiments in Aim 1 will characterize the heterogeneity in cargo release from activated platelets. The goal is to determine if platelet secretion is thematic, stochastic, or controlled kinetically. Micro-ELISA assays will be used to measure the secretion of several released molecules and detailed mathematical analysis of the release kinetics will be used to identify patterns. The second Aim seeks to dissect a novel signaling cascade that directly controls platelet secretion. The goal is to identify the signaling pathways that activate IkB Kinase to phosphorylate SNAP-23 and to determine how the modification affects platelet secretion. This Aim focuses on the role that IkB Kinase (IKK) plays in regulating platelet secretion by probing both how the kinase is activated and how SNAP-23 phosphorylation affects platelet secretion. Additional studies will determine if IKK inhibitors can be used as anti-thrombotic agents. The final Aim focuses on the mechanism of a limiting element of the platelet secretory machinery, Munc13-4. The goal will be to define the mechanism by which Munc13-4 mediates platelet secretion. The experiments in this Aim will characterize the molecular mechanisms of Munc13-4 by probing its interactions and their roles in secretion. Since Munc13-4 is a limiting factor in platelets and is required for normal hemostasis, it is an attractive target for therapeutic intervention. The experiments in the final Aim will yield the detailed molecular characterization required for such targeting. In sum, the data gained for the proposed experiments will illuminate the process of platelet secretion and thus set the constraints for future models of platelet function in the vasculature. The experiments will also identify aspects of the secretory machinery that can be exploited for therapeutic intervention to modulate thrombosis. Cardiovascular disease is the #1 cause of death in the US and is estimated to cost $500 billion in medical expenses and lost productivity in 2010 (AHA Fact Sheet). These staggering statistics underline the need for new therapeutic agents designed to ameliorate the pathologies associated with platelet dysfunction. The insights gained from the proposed experiments and their potential for identifying novel targets will clearly benefit the search for such agents. PUBLIC HEALTH RELEVANCE: Strokes and heart attacks are major killers in the United States and both are caused by a loss of blood flow, usually because a blood clot has formed in the wrong place. This application focuses on how platelets release molecules that cause clot formation. This work is critical because it will direct the development of future drugs needed to decrease the risk of spurious clot formation.